Rapamycin-induced cytotoxic signal transduction pathway.
We examined the effects of rapamycin on activation, proliferation, and expression of cytotoxic effector molecules in Molt-4 human T lymphocytes. We investigated the effects of rapamycin on cell viability, caspase family protein activities. Western blots of Bcl-2, Bak, p53, p21, p27, Rb, CDK2, and cyclin B1, as well as measurement of reactive oxygen species (ROS) generation and mitochondrial membrane potential transition. Cells were cultured in the presence or absence of rapamycin. Flow cytometric analysis was performed using propidium iodide stain. Viability of Molt-4 cells was decreased by the addition of rapamycin in dose- and time-dependent manners. Rapamycin induced no nuclear fragmentation in Molt-4 cells. Generation of H2O2 in rapamycin-treated Molt-4 cells increased in a time-dependent manner. There were no changes among catalytic activities of caspase proteases. And there was no evidence of expression of Bcl-2, p53, p21, p27, or Rb proteins. G2/M phase cell cycle arrest was identified by flow cytometry. We noted decreased expressions of CDK2 and cyclin B1. We also noted increased Bak protein expression and change in mitochondrial membrane potential transition. In conclusion, rapamycin-induced cytotoxicity was characterized by generation of ROS, which modulated Bak protein expression and mitochondrial dysfunction. G2/M phase cell cycle arrest was achieved by decreased expressions of CDK2 and cyclin B1.
Choi
S J N
SJ
Division of Transplantation Surgery, Department of Surgery, Chonnam National University Medical School, Gwangju, Korea. choisjn@chonnam.ac.kr
You
H S
HS
Chung
S Y
SY
D000199:Actins / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects*; D005434:Flow Cytometry; D006801:Humans; D007166:Immunosuppressive Agents / Q000633:toxicity; D008564:Membrane Potentials / Q000187:drug effects; D051336:Mitochondrial Membranes / Q000187:drug effects / Q000502:physiology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D020123:Sirolimus / Q000494:pharmacology*; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology*; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000187:drug effects
D000199:Actins; D007166:Immunosuppressive Agents; D019253:Proto-Oncogene Proteins c-bcl-2; D050998:bcl-2 Homologous Antagonist-Killer Protein; D020123:Sirolimus